1. Field of the Invention
This invention relates generally to the field of gene therapy and more particularly to the production and use of defensin polypeptide zinc finger-nucleotide binding and linker motifs.
2. Description of Related Art
Defensins are cationic, cysteine-rich peptides that display broad spectrum antimicrobial activity. Their structure is characterized by a conserved cysteine motif that forms three disulfide linkages, imposing a characteristic β-sheet structure (Hill et al., 1991; White et al., 1995). Associated with this structure is an amphiphilic charge distribution that enables the defensins to interact with and disrupt target cell membranes (Lehrer et al., 1989). This disruption is thought to be accomplished by the formation of channels in the target membrane, leading to cell lysis (Kagan et al., 1990). Defensins have been shown to inhibit proliferation of both gram-positive and gram-negative bacteria, yeast and numerous viruses. In particular, defensins inhibit the proliferation of the yeast strain Candida albicans and the gram-negative bacteria Escherichia coli (Porter et al., 1997; Harder et al., 1997; Schonwetter et al., 1995; Daher et al., 1986).
Defensins have recently been identified as an integral component of the antimicrobial barrier of mucosal surfaces. In both the human and murine small intestine, defensin RNA has been localized to the Paneth cell, a specialized epithelial cell located at the crypt base (Ouellette et al., 1989; Jones et al., 1992). The associated peptide has been localized within secretory granules of the Paneth cell and in the lumen of the small intestine, suggesting a role for defensins in host defense in the gut (Selsted et al., 1992). Defensins have also been found in bovine and human respiratory epithelium. Tracheal antimicrobial peptide, a β-defensin isolated from bovine tracheal mucosa, was localized to the ciliated columnar epithelial cells of the trachea and bronchi (Diamond et al., 1991; Diamond et al., 1993). Lingual antimicrobial peptide, another β-defensin, was found in bovine lingual mucosa and stratified squamous epithelium of the tongue (Schonwetter et al., 1995). Most recently, human β-defensin-1 was demonstrated to be present in the epithelium of the trachea and bronchi, as well as the submucosal gland and alveolar epithelium (Goldman et al., 1997; Zhao et al., 1996).
Considerable data exists indicating that epithelial defensins are up-regulated in response to infection. In cultured tracheal epithelial cells, tracheal antimicrobial peptide message is induced following exposure to bacterial lipopolysaccharide (Diamond et al., 1996). This induction was blocked by antibody to CD14, suggesting that epithelial cells provide an active, inducible antimicrobial defense. Following injury to bovine tongue, lingual antimicrobial peptide RNA message increased at the site of injury (Schonwetter et al., 1995). Induction of lingual antimicrobial peptide was also observed following acute infection in bronchial epithelium and chronic infection in ileal mucosa (Stolzenberg et al., 1997). Together these data support a role for β-defensins as important host defense effector molecules that are rapidly mobilized by epithelium upon injury or infection.
Due to the significant host defense properties of defensins, any means which stimulates or induces the production of these peptides is desired.
The foregoing discussion of the prior art is taken largely from published U.S. Patent Application No. 2002/0076393 to Fehlbaum et al., who propose a method of increasing the production of defensins in eukaryotic cells by exposing the eukaryotic cells to a composition comprising isoleucine or active isomers or analogs thereof in an amount sufficient to effect the desired increase.